The successes of proteasome inhibitors bortezomib (Velcade(R)) and carfilzomib (Kyprolis(R)) as FDA-approved cancer drugs have invigorated efforts to devise new cancer therapeutics targeting upstream pathways involving enzymes of ubiquitin and ubiquitin-like protein modification systems. Our long-term goal is to provide therapies that inhibit the NEDD8 system and the major NEDD8 targets cullin-RING ubiquitin ligases to directly benefit cancer patients. The NEDD8-activating enzyme (NAE) inhibitor MLN4924 is currently the most advanced of these potential new classes of drugs. MLN4924 has extensive published pre-clinical data demonstrating strong anti-cancer efficacy and several Phase 1 clinical trials are in progress for hematologic and advanced non-hematologic malignancies. Given the clear benefit MLN4924 provides by selectively inducing cancer cell death with no noted toxicities or side-effects on experimental cancers, it remains highly significant to understand how cells respond to NEDD8 system inhibition. As an important step towards this goal and in response to PAR-12-145 NCI Exploratory/Developmental Research Grant Program, the overall objective of this proposed study is to use insight derived from experiments examining on-target MLN4924 resistance to identify new NAE inhibitors that function through mechanisms of action distinct from MLN4924. Through our strong preliminary data, we have devised a highly innovative strategy that uniquely positions us to describe the types of on-target mutations possible for MLN4924 resistance. We have also generated a new screening assay based on differential scanning fluorimetry. This uses a ligand competition strategy to evaluate the mechanism of action of molecules that bind NAE. Our central hypothesis is that the diversity of biochemical changes in NAE sufficient for cancer cell resistance to MLN4924 can be overcome by small molecules that inhibit NAE through mechanisms not involving the enzyme's catalytic pocket. To test this hypothesis and achieve the overall objective of the research, two Specific Aims are proposed: 1) to characterize the biochemical diversity of MLN4924-resistant forms of NAE and 2) to identify small molecule inhibitors of these drug-resistant NAE complexes. IMPACT: This work is highly innovative with direct translational relevance due to the extraordinary opportunity it provides to describe MLN4924 resistance mechanisms in detail before they are reported in relapsed patients and to use this information to discover new NAE inhibitors that function through distinct mechanisms of action. Collectively, these efforts will significantly contribute to providing much needed new treatment options for cancer patients.